天然抗氧化物经由抗硝化应激机制介导缺血性脑损伤保护作用研究

发布时间：2018-06-26 00:45

本文选题：脑卒中 + SAA　；参考：《浙江大学》2017年博士论文

【摘要】：在世界范围内,脑卒中是一种伴有较高发病率和致死率的常见健康问题,它在发展中国家的发生率呈快速上升趋势。世界卫生组织预估,每年有1500万人新发脑卒中,70%伴有残疾甚至死亡。在老龄人口中,脑卒中导致的死亡率高达三分之二,是第二大致死性疾病。近年来,在中国等发展中国家中由于生活方式的改变,脑卒中的发病率呈现上升趋势,我们亟需了解脑卒中的潜在发病机制并发展新的治疗方法。在脑血管病的发展进程中,活性氮及硝化应激(Nitrosative stress)参与介导细胞损伤。缺血性脑损伤引起脑部血液供应中断,导致缺氧和低糖,以致神经元无法维持细胞稳态和基本功能所需的离子浓度梯度。研究表明,在缺血的脑组织中,谷氨酸含量在缺血发生后快速上升。过量激活NMDA谷氨酸受体并导致钙离子(Ca2+)浓度上升,Ca2+的释放会使各种信号通路激活最终损伤神经元功能甚至引起细胞死亡。Ca2+介导钙调蛋白(CaM)和一氧化氮合酶(NOS)的偶联,改变内皮型一氧化氮合酶(eNOS)的功能。阻断eNOS的功能会因为NADPH氧化酶的激活而引起超氧根离子(O2-·)浓度上升。一氧化氮(NO)和O2-·浓度上升,最终反应生成过氧亚硝基阴离子(ONOO-)。以过氧亚硝基阴离子为代表的活性氮形成的硝化应激,会影响包括血管舒张,神经传递,免疫防御和细胞运动性调控等多种细胞生理过程。硝化应激与多种疾病发生发展进程相关,它对细胞结构的损伤包括脂类,细胞膜,蛋白质和DNA。大脑脉管系统是危险因素下硝化应激介导的缺血性脑损伤病理过程中的重要靶组织。因此,靶向硝化应激的神经保护策略可能对脑卒中的治疗具有积极的作用。在本研究中,我们分别使用了两种天然强效抗氧剂——丹酚酸A(SAA)和β-胡萝卜素(BC)。利用小鼠短暂性中脑动脉闭塞(tMCAO)模型,考察它们保护缺血性脑损伤的药理学作用及潜在分子机制。在中药相关文献中,SAA被称为丹参。SAA是一种多靶点药物,抑制多种缺血损伤因子所激活的胞内信号通路。SAA是从中药丹参中提取而得,已有文献报道,它对心脑血管疾病有治疗作用。丹参根和茎的切片经薄层色谱分析,柱色谱提纯,高效液相色谱制备粗产。我们考察了 SAA抗神经血管损伤的药理学效应,此外也探索了其药理作用与激活钙蛋白酶和eNOS脱偶联之间的关系。C57BL/6雄性小鼠在中脑动脉梗阻60min模型建立前连续七天给予腹腔注射SAA(1 mg/kg和5mg/kg)或生理盐水作为对照。高分辨率正电子发射断层(PET)扫描仪可用于小型动物成像以检测18F-FDG葡萄糖在脑内的代谢,用于再灌注24小时后药物改善脑代谢功能的检测;再灌注23h后,转棒实验及神经功能学评分实验用于评估小鼠功能性行为;脑梗阻程度采用Nissl染色进行考察。钙蛋白水解酶的活性,eNOS脱偶联以及其它细胞凋亡级联反应关联蛋白也作为检测SAA抗神经血管损伤的重要指标。相较于模型组,SAA(1 mg/kg和5 mg/kg)预处理的组可有效减轻神经功能障碍。在旋转实验中,SAA处理组相较于模型组跌倒倾向明显降低。Nissl染色脑片显示模型组存在大面积脑梗塞,而SAA(1 mg/kg和5 mg/kg)处理组脑梗塞面积明显有所减少。PET结果提示,SAA预处理可以有效提高模型组葡萄糖的代谢率。相较于假手术组,模型组小鼠脑内缺血侧相较于对照侧区域的摄取值的比值明显减少,而SAA预处理可有些改善两侧的差异。此外,SAA可以抑制钙蛋白酶的水解能力。血影蛋白Spectrin的裂解量间接反映了钙蛋白酶的活性。蛋白质免疫印迹分析显示,相较于假手术组,缺血侧脑组织的血影蛋白崩解片段明显增多。用SAA(1 mg/kg和5 mg/kg)处理可以有效减轻保护血影蛋白崩解片段的产生。脑卒中也会加强钙蛋白酶介导的钙调磷酸酶(CaN)的激活,SAA(1 mg/kg和5 mg/kg)处理同样也会抑制钙蛋白酶介导的CaN降解。此外,eNOS二聚体的形成可以产生生理效应的NO,然而单体的形成则作为eNOS脱偶联的标志物,可促进OO-生成。给予SAA(1 mg/kg和5 mg/kg)后,可以显著减少tMCAO后eNOS二聚体和单体的比率,具有一定量效关系。在免疫印迹分析中,我们观察到模型组缺血脑区AKT,ERK和FKHR的去磷酸化现象。我们推测磷酸化水平的下降可能是由eNOS脱偶联引起的O2-·上升及硝化应激导致的。SAA预处理(1 mg/kg和5 mg/kg)能显著提高AKT,ERK和FKHR的磷酸化水平。我们的实验结果显示SAA具有较好的抗缺血性神经损伤作用。SAA给药提高缺血小鼠脑葡萄糖的代谢及抗神经功能障碍与其抑制钙蛋白酶的激活、抑制eNOS脱偶联及硝化应激损伤相关,从而保护大脑免遭缺血损伤。β-胡萝卜素(BC)是一种具有抗氧化功能的重要类胡萝卜素。β-胡萝l卜素因具有抗氧化功能而降低多种心脏和脑部疾病的风险,有报道称低剂量的β-胡萝卜素可降低心肌缺血的发病风险。血脑屏障可以阻挡大量的中枢神经系统激动剂。过去几十年,纳米粒子在中枢神经系统给药领域占据重要地位,纳米给药系统有加速药物穿过BBB的功能。脑损伤靶标分子的修饰可帮助纳米给药系统靶向到特定损伤区域,本实验考察利用硝基酪氨酸抗体修饰给药系统的靶向性。实验中,我们采用了三种β-胡萝卜素制剂,包括提高β-胡萝卜素溶解度的乳剂(BC-EM),BC偶联PEG修饰的脂质纳米粒(BC-PLNs),抗3-硝基酪氨酸抗体修饰的BC偶联PEG修饰的脂质纳米粒(BC-NT/PLN)。实验分为七个组,包括假手术组,模型组,BC-EM(10 mg/kg)组,BC-PLNs(2.5 mg/kg)组,BC-NT/PLNs(0.625 mg/kg,1.25mg/kg 或 2.5 mg/kg)组。MCAO模型建立后 60 min,根据小鼠体重按比例静脉注射药物或药物载体。在再灌注23小时末估算神经功能损伤程度,并取出大脑样本进行梗阻损伤区域的Nissl染色。通过脑片与荧光探针NP3孵育的结果评价ONOO-的产生量。免疫印迹技术可见钙蛋白酶的激活,紧密连接蛋白,phospho-AKT(Ser 473),phospho-FKHR(Ser256)和 phospho-ERK(Thr202/Tyr204)的磷酸化。BBB 的完整性由伊文氏蓝对组织溢出物染色来鉴定。我们发现相较于空白组,BC-EM对神经损伤有明显的修复作用。而相较于BC-EM,BC-PLNs在极低浓度时就能显示神经保护功能。2.5 mg/kg剂量的BC-PLNs在减少神经损伤和缩小梗阻区域方面显示出特有的优势。但是,为了达到治疗脑卒中的目的,我们要将附有3-硝基酪氨酸抗体的BC靶向到缺血区域。在中脑动脉梗阻和再灌注时相中有大量ONOO-产生。相较于空白组,低剂量(0.625mg/kg或1.25 mg/kg)被NT/PLNs偶联的BC即可对功能损伤有所改善,而2.5 mg/kg的剂量对损伤没有显著改变。同时,静注低剂量(0.625mg/kg或1.25 mg/kg)被3-NT/PLNs修饰的BC有效减少MCAO模型中的梗阻区域。NT/BC/PLNs在低剂量时具有神经保护作用并且能对抗脑缺血引起的损伤。我们的结果显示,BC-EM(10 mg/kg),PLNs 包载 2.5 mg/kg BC 和 0.625mg/kg 3NT/PLNS能有效激活钙蛋白酶并抑制血影蛋白和CaN的降解。我们发现,给予BC-EM(10 mg/kg)可有效抑制 phospho-P38(Thr180/Tyr182)和 phospho-JNK(Thr183/Tyr185)的上调。缺血会引起许多蛋白 phospho-AKT(Ser 473),phospho-FKHR(Ser256)和 phospho-ERK(Thr202/Tyr204)磷酸化水平的下降。NT/BC/PLNs(0.625mg/kg)有效抑制上述蛋白质磷酸化水平的下调。过氧亚硝基阴离子(ONOO-)是一种对组织有损伤作用的强氧化剂和强硝化剂,硝化应激在缺血过程中能导致脑微血管损伤及破坏神经血管单元。NP3作为一种对ONOOC敏感的荧光探针,孵育使用后,相比于对照组,模型组大脑受损侧局部微血管处显示极强的NP3荧光强度,提示ONOO-浓度出现显著上升。NT/PEG纳米结合物(0.625mg/kg)有效抑制硝化应激并保护微血管受损。利用受损的血管中渗漏出的伊文氏蓝可以判断微血管损伤程度。在空白组中,MCAO 24小时后,伊文氏蓝溶液从血管渗入脑实质的量大大提高。相比之下,NT/PEG-纳米结合物(0.625mg/kg)组则有效减少伊文氏蓝的渗漏并且抑制了紧密连接蛋白ZO-1和闭合蛋白Occludin的退化。相较于常规β-胡萝卜素乳剂,较低剂量的BC脑靶向递药系统NT/BC/PLNs有效地改善了脑损伤。利用抗体偶联策略,NT/BC/PLNs递药系统将β-胡萝卜素靶向输送到ONOO-生成的脑缺血损伤区域。此外,该系统减少ONOO-的生成,并且抑制伊文氏蓝渗漏,降低微血管的破损。同时通过抑制钙蛋白酶的激活,减少缺血损伤中血影蛋白和CaN的降解,最终起到保护神经血管单元作用。综上所述,硝化应激参与缺血后脑损伤病理过程。丹酚酸A和β-胡萝卜素具有一定的抗缺血性脑损伤作用,其分子机制与减轻eNOS脱偶联和硝化应激损伤相关联。此外,我们发现偶联Nitrotyrosine抗体的脑靶向纳米给药系统不仅可以定向输送药物到缺血脑区,同时也可以减少负载药物剂量起到同等治疗作用。
[Abstract]:In the world, stroke is a common health problem with high morbidity and mortality, and its incidence in developing countries is rising rapidly. The WHO estimates that 15 million people have new stroke, 70% with disability or even death every year. The mortality rate of stroke is up to 2/3 in the elderly population. In recent years, the incidence of stroke is rising in China and other developing countries, in China and other developing countries. We need to understand the potential pathogenesis of stroke and develop new treatment methods. In the development of cerebrovascular disease, active nitrogen and nitrification stress (Nitrosative stress) participate in the development of cerebrovascular disease. Mediating cell damage. Ischemic brain damage causes disruption of the blood supply of the brain, resulting in hypoxia and low sugar, so that neurons are unable to maintain the ionic concentration gradient required for cell homeostasis and basic function. The release of ions (Ca2+) increases, and the release of Ca2+ activates the function of the signaling pathway to eventually damage the neuron function and even causes the cell death.Ca2+ to mediate the coupling of calmodulin (CaM) and nitric oxide synthase (NOS), and changes the function of the endothelial nitric oxide synthase (eNOS). The inhibition of the function of eNOS causes SUPEROXYGEN due to the activation of NADPH oxidase. The concentration of the root ion (O2-) rises. The concentration of nitric oxide (NO) and O2- increases, and the final reaction produces peroxy nitroso anion (ONOO-). Nitrification stress formed by the active nitrogen, represented by the peroxy nitroso anions, will affect a variety of cell physiological processes, including vasodilatation, neurotransmission, immune defense and cell motility regulation. Stress is associated with the development of a variety of diseases. Its damage to cell structures, including lipids, cell membranes, protein and DNA. cerebral vascular system, is an important target tissue in the pathological process of ischemic brain damage mediated by nitrification stress. Therefore, the neuroprotective strategy targeted to nitrification stress may be used to treat stroke. In this study, two natural strong antioxidants, salvianolic acid A (SAA) and beta carotene (BC), were used respectively. The pharmacological action and potential molecular mechanism of the mouse transient middle cerebral artery occlusion (tMCAO) were used to investigate the protection of ischemic brain damage. In the related literature of Chinese medicine, SAA was called Salvia miltiorrhiza. .SAA is a multi target drug. The intracellular signaling pathway,.SAA, which is activated by a variety of ischemic injury factors, is extracted from the traditional Chinese medicine Salvia miltiorrhiza. It has been reported in the literature. It has a therapeutic effect on cardiovascular and cerebrovascular diseases. The slices of root and stem of Salvia miltiorrhiza are analyzed by thin layer chromatography, purified by column chromatography and high performance liquid chromatography to produce crude production. We have investigated SAA The pharmacological effects of anti neurovascular injury, and the relationship between its pharmacological effects and the activation of calsin and eNOS deactivation were also explored..C57BL/6 male mice were given SAA (1 mg/kg and 5mg/kg) or physiological salt water for seven days before the establishment of 60min model of middle cerebral artery obstruction. PET) the scanners can be used for small animal imaging to detect the metabolism of 18F-FDG glucose in the brain and to improve the detection of brain metabolism after 24 hours of reperfusion. After reperfusion of 23h, the rotation rod experiment and neurologic score test are used to evaluate the functional behavior of mice; the degree of cerebral infarction is examined by Nissl staining. Enzyme activity, eNOS decoupling and other cell apoptosis cascade reaction proteins are also important indicators for detecting SAA's neurovascular damage. Compared to model groups, SAA (1 mg/kg and 5 mg/kg) pretreated groups can effectively reduce neural dysfunction. In the rotation experiment, the group of SAA is prone to lower.Nissl compared to the model group falling down. Dyed brain slices showed that there was a large area of cerebral infarction in the model group, while the area of cerebral infarction in SAA (1 mg/kg and 5 mg/kg) treatment group was significantly reduced by.PET, and SAA preconditioning could effectively improve the metabolic rate of glucose in the model group. Compared to the sham group, the ratio of the blood deficiency in the model group to the control side of the control group was obvious. SAA pretreatment could significantly reduce the difference between the two sides. In addition, SAA could inhibit the hydrolysis of calpain. The fragmentation of HP Spectrin reflects the activity of calpain indirectly. Protein immunoblotting analysis showed that the disintegration fragment of the ischemic side group was significantly increased compared with the sham group. SAA (1 m) G/kg and 5 mg/kg) treatment can effectively reduce the production of the protection of the disintegration fragments. Stroke also enhances calprotease mediated calcineurin (CaN) activation. SAA (1 mg/kg and 5 mg/kg) can also inhibit calprotease mediated CaN degradation. In addition, the formation of eNOS two polymers can produce physiological effects of NO, but monomers The formation of eNOS, as a marker for eNOS decoupling, can promote OO- formation. After giving SAA (1 mg/kg and 5 mg/kg), the ratio of eNOS two polymers and monomers can be significantly reduced after tMCAO. In Western blot analysis, we observed the dephosphorylation of AKT, ERK and FKHR in the ischemic brain area of the model group. We speculate phosphorylated water. The decrease of the level may be the.SAA preconditioning (1 mg/kg and 5 mg/kg) induced by eNOS dehydration and nitrification stress (1 mg/kg and 5 mg/kg). Our experimental results show that SAA has a better anti ischemic nerve damage effect and.SAA gives the metabolism and anti nerve of glucose in the brain of high ischemic mice. Dysfunction is related to the inhibition of calsin activation, inhibition of eNOS decoupling and nitrification stress damage, which protects the brain from ischemic injury. Beta carotene (BC) is an important carotenoid with antioxidant function. Beta carotene L dopin has the risk of reducing various heart and brain diseases because of its antioxidant function. The low dose of beta carotene can reduce the risk of myocardial ischemia. The blood brain barrier can block a large number of central nervous system agonists. Nanoparticles have played an important role in the central nervous system for the past few decades. The nano drug delivery system has the function of accelerating the drug passing through the BBB. The modification of the target molecules of the brain damage can help. In this experiment, we used three beta carotene preparations, including BC-EM, BC coupled with PEG modified lipid nanoparticles (BC-PLNs), and the anti 3- nitrotyrosine antibody repair. BC coupled PEG modified lipid nanoparticles (BC-NT/PLN). The experiment was divided into seven groups, including sham operation group, model group, BC-EM (10 mg/kg) group, BC-PLNs (2.5 mg/kg) group, BC-NT/PLNs (0.625 mg/kg, 1.25mg/kg, or 2.5 mg/kg) group.MCAO model after establishment 60. The root was injected intravenously or drug carrier according to the weight of mice. In the reperfusion 23 The degree of nerve function damage was estimated at the end of the hour, and the brain samples were taken out for Nissl staining in the area of the obstruction. The production of ONOO- was evaluated by the incubation of brain slices with the fluorescent probe NP3. The immunoblotting technique showed the activation of calsin, the close connexin, phospho-AKT (Ser 473), phospho-FKHR (Ser256) and phospho-ERK (Thr202/T). The integrity of phosphorylated.BBB in yr204 was identified by the staining of eviner blue against tissue spillovers. We found that BC-EM had a significant repair effect on nerve damage compared to the blank group. Compared to BC-EM, BC-PLNs at very low concentrations could show the BC-PLNs of the neuroprotective function of.2.5 mg/kg in reducing nerve damage and narrowing the area of obstruction. The domain shows a unique advantage. However, in order to achieve the purpose of treating stroke, we should target the BC with 3- nitrotyrosine antibody to the ischemic region. There are a large number of ONOO- in the middle cerebral artery obstruction and reperfusion. Compared to the blank group, the low dose (0.625mg/ kg or 1.25 mg/kg) is NT/PLNs coupled BC to the function. The damage was improved, and the dose of 2.5 mg/kg did not change significantly. At the same time, intravenous injection of low dose (0.625mg/kg or 1.25 mg/kg) by 3-NT/PLNs modified BC effectively reduced the.NT/BC/PLNs in the MCAO model, which had a neuroprotective effect at low doses and could antagonize the damage caused by cerebral ischemia. Our results showed that BC-EM (10) Mg/kg), PLNs encapsulation of 2.5 mg/kg BC and 0.625mg/kg 3NT/PLNS effectively activates calsin and inhibits the degradation of spectrin and CaN. We have found that BC-EM (10 mg/kg) can effectively inhibit the up regulation of phospho-P38 (Thr180/Tyr182) and phospho-JNK (473). R256) and the decrease of phosphorylation level of phospho-ERK (Thr202/Tyr204).NT/BC/PLNs (0.625mg/kg) effectively inhibit the downregulation of the protein phosphorylation level. Peroxiso nitroso anion (ONOO-) is a strong oxidant and strong nitrifying agent that has damage to tissue. Nitrification should induce cerebral microvascular damage and destruction of the brain during ischemia. The vascular unit.NP3 was used as a ONOOC sensitive fluorescent probe. After incubation, compared to the control group, the local microvessels of the brain damaged side of the model group showed a strong NP3 fluorescence intensity, suggesting a significant increase in the concentration of ONOO- in the.NT/PEG nanoscale (0.625mg/kg) to inhibit nitrification stress and protect the microvascular damage. In the blank group, the amount of even's blue solution infiltrated from the blood vessels to the parenchyma significantly increased in the blank group. In contrast, the NT/PEG- nanocombination (0.625mg/kg) group reduced the leaking of even blue and suppressed the close connexin ZO-1 and closed eggs in the 0.625mg/kg group. The degradation of white Occludin. Compared to the conventional beta carotene emulsion, the lower dose of the BC brain targeting delivery system NT/BC/PLNs effectively improves brain damage. The NT/BC/PLNs delivery system uses the antibody coupling strategy to deliver beta carotene targeting to ONOO- generated ischemic injury areas. In addition, the system reduces the formation of ONOO- and inhibits the formation of the beta carotene. Evans blue leaks and reduces the damage of microvessels. At the same time, by inhibiting the activation of calsin, reducing the degradation of blood shadow protein and CaN in ischemic injury, and ultimately protecting the neurovascular unit. To sum up, nitrification stress participates in the pathological process of cerebral injury after ischemia. The salvianolic acid A and beta carotene have certain anti ischemic brain damage. The molecular mechanism of injury is associated with the reduction of eNOS decoupling and nitrification stress damage. In addition, we found that the brain targeted nano drug delivery system coupled with Nitrotyrosine antibodies can not only deliver drugs to the ischemic brain area, but also reduce the dose of the loaded drug to the same therapeutic effect.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285

【参考文献】